T-lymphocytes are able to interact in a specific manner with a wide variety of antigens through highly diverse recognition structures known as T-cell receptors (TcRs). These heterodimers are composed of alpha and beta chains which consist of variable (V) and constant (C) regions homologous to those of antibodies. In contrast to antibodies, however, which recognize antigen in intact form, TcRs recognize antigen only as peptide fragments bound to molecules of the major histocompatibility complex (MHC). In addition, TcRs interact with a class of molecules known as 'superantigens' which can induce profound immunological responses in the host such as toxic shock and the deletion of particular T-cell populations during ontogeny. While much is known about the three-dimensional structure of antibodies and antigen-antibody complexes, no direct structural information is available on TcRs. We propose to investigate the molecular basis of antigen recognition by these molecules through determination of their three-dimensional structure in free and liganded forms using X-ray crystallographic techniques. We have crystallized the extracellular portion of the beta chain of a TcR (designated 14.3.d) specific for a hemagglutinin peptide (HA 110-120) of influenza virus in association with the MHC class II I-E(d) molecule and have now determined its three- dimensional structure to 2.4 Angstrom resolution. We have also crystallized a bacterially produced V-alpha domain from a TcR specific for the N-terminal nonapeptide of myelin basic protein (a self antigen) in the context of I-A(u); these crystals diffract to beyond 2.2 Angstrom resolution. Determination of the three-dimensional structure of these molecules will enable us to define the basic architecture of both TcR V subunits and to construct a model of an associated alpha-beta heterodimer in advance of an actual structural determination. This work will provide the basis for detailed structure-function studies of TcRs with their physiological ligands. We will first characterize the binding in solution of the 14.3.d alpha-beta TcR to various staphylococcal enterotoxins and to its cognate peptide/MHC complex. For the latter purpose, we will use a soluble form of I-E(d) which can be loaded with analogs of HA 110-120 to enable us to map TcR-peptide contacts. We will also seek to isolate variants of the antigenic peptide from a phage display library able to bind the l4.3.d TcR with sufficient affinity in the absence of MHC to permit crystallization of peptide-TcR complexes. Finally, we will attempt to crystallize complexes between the l4.3.d TcR and superantigens and between this TcR and its specific peptide/MHC ligand. These studies will significantly advance our understanding of antigen recognition T-cells and contribute to the design of therapeutic agents for regulating T-cell responses to foreign and self antigens.